The interferon antiviral defense program is activated by the host innate immune system upon sensing RNA virus infection. However, many RNA viruses have evolved mechanisms to subvert this defense program. The underlying host-viral interactions that dictate the outcome of viral infection, including the molecular mechanisms that regulate antiviral innate immune signaling to drive an effective immune response to virus infection, are not fully understood. An understanding of these processes is required to define the critical components that drive the first line of host defense to RNA virus infection. The hepatitis C virus (HCV) NS3/4A protease cleaves the host antiviral signaling proteins Riplet and MAVS to block this antiviral signaling. Preliminary studies have identified a mutation in NS3/4A that inhibits Riplet cleavage while maintaining MAVS cleavage. Our studies using this mutant HCV have found that the E3 ubiquitin ligase Riplet regulates a signaling pathway independent of the known pattern recognition receptor and antiviral protein RIG-I that is essential for innate immunity to HCV. Therefore, the goal of this proposal is to define this Riplet-dependent, RIG-I independent antiviral signaling pathway. Based on our preliminary data, the central hypothesis of this proposal is that Riplet ubiquitinate a protein that drives a RIG-I independent antiviral signaling pathway that is regulated during HCV infection to control the antiviral response and outcome of infection. Guided by our preliminary data, this hypothesis will be tested by pursuing the following two specific aims: 1) Define the RIG-I independent innate immune signaling pathway regulated by Riplet; 2) Identify the targets of Riplet ubiquitination during HCV infection. In Aim 1, the molecular mechanisms by which this RIG-I independent signaling pathway regulated by NS3/4A contributes to innate immune signaling and the antiviral response will be defined. In Aim 2, the specific ubiquitinated target(s) of Riplet will be identified and validated. Taken together, the work proposed in this application will be significant because it will define a new antiviral innate immune signaling pathway that will have implications for the treatment and prevention of RNA virus infection and interferon-mediated autoimmune disease. The proposed work is innovative because it is the first to uncouple distinct innate immune regulatory mechanisms within the HCV NS3/4A protein that will be used to fully define the roles of Riplet and MAVS in the antiviral response and to uncover a new Riplet-regulated antiviral signaling pathway. Ultimately, an increased understanding of the regulation of innate immune pathways will improve our knowledge of the mechanistic causes of dysregulated interferon production that can lead to autoimmune disease, and it will define the mechanisms of immune protection for RNA virus infection that will have implications for therapeutic and vaccine strategies to limit RNA virus infection.